Abstract LB-355: A novel non-COX-inhibitory sulindac derivative induces autophagic cell death in lung adenocarcinoma cells

Abstract

Abstract Autophagy is a normal catabolic process, which serves to remove damaged organelles and long-lived proteins within the cell. Several studies have provided evidence that some tumor cell lines respond to chemotherapeutic agents by triggering autophagy and inducing autophagic cell death (or programmed cell death type II) characterized by the formation of autophagic vacuoles in the cytoplasm. This, along with observations that autophagic activity and signaling are reduced during malignant transformation suggest that induction of autophagic cell death may have a therapeutic value in cancer. In this study, we describe a novel non-COX inhibitory derivative of NSAID sulindac, referred to as sulindac sulfide amide (SSA), which was found to inhibit the growth of lung adenocarcinoma cell lines primarily through the induction of autophagy with or without apoptosis. SSA inhibited the growth of human lung tumor cells (A549, H1299, HOP-62) in a dose-dependent manner with IC50 values of 3-5 µM, while sulindac sulfide inhibited growth with IC50 values of 50-60 µM. In addition, SSA treatment led to a G0/G1 arrest and decreased proliferation as measured by EdU incorporation. SSA induced apoptosis of A549 lung tumor cells as determined by caspase 3/7 and AnnexinV assays, however, caspase-induction was absent in H1299 cells suggesting an alternative mechanism of cell death. Interestingly, staining SSA-treated A549 cells with Lyso-ID green dye revealed a dramatic increase in the number and size of acidic vesicles within the cytoplasm. Moreover, histological analysis of dying cells demonstrated multiple, large intracellular vacuoles consistent with autophagy. Western blotting for autophagosome-associated LC3 protein demonstrated that SSA induces a dose- and time-dependent increase in autophagic vacuole formation. This was confirmed by transiently transfecting A549 cells to overexpress the LC3 protein fused to the enhanced green fluorescent protein. 5μM SSA treatment for 24h significantly increased the number of cells with LC3-positive vesicles as well as the number of vesicles within the cytoplasm. These effects were not observed with sulindac sulfide, which suggests a distinct mechanism of action. SSA inhibited the phosphorylation of Akt and its downstream effectors mTOR, 4EBP and p70S6K resulting in induction of autophagy. The expression of AMPK, an inhibitor of mTOR function, was shown to be upregulated while levels of cyclinD1 and survivin, important mediators of tumor cell proliferation and death respectively, were downregulated. Our results suggest that SSA can induce autophagy by a mechanism involving the suppression of Akt/mTor signaling. We conclude that SSA is a highly promising chemopreventive or chemotherapeutic agent due to its ability to induce both caspase-dependent apoptotic and caspase-independent autophagic cell death. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-355. doi:1538-7445.AM2012-LB-355